Fruits, stems, and leaves change color in response to developmental and environmental factors. Although these tissues have very different functional, morphological, and biochemical attributes their cells undergo similar changes in ultrastructure and chloroplast composition during fruit ripening and leaf senescence. Chlorophyll breakdown is a primary biochemical event in color changes and the first step in the chlorophyll degradation pathway is catalyzed by chlorophyllase (EC 3.1.1.14). This enzyme catalyzes the hydrolysis of the phytol chain in chlorophylls or pheophytins to produce chlorophyllides or pheophorbides. Chlorophyllases of different mobilities on polyacrylamide gels have been purified from plants and algae and in some instances, like in citrus, there are two chlorophyllase bands present. It is not known what the difference in size represents.
Genes encoding chlorophyllases have been purified from Valencia oranges and Chenopodium album. The Valencia orange Chlase1 gene was isolated from a library prepared from mRNA extracted from the fruit peel, stems and leaves. The steady state level of Chlase1 mRNA increased with ethylene treatment (Jacob-Wilk, et al. Plant J. (1999) 20:653–661). Although three proteins, of different molecular weight, with chlorophyllase activity have been isolated from Chenopodium album, only the gene encoding one of them (CaCLH C. alum chlorophyll-chlorophyllido hydrolase) has been identified (Tsuchiya et al. (1999) Proc. Natl. Acad. Sci. USA 96:15362–15367). Using sequence similarity to the C. album CaCLH chlorophyllase sequence two Arabidopsis thaliana genes have also been assigned chlorophyllase activity. Tsuchiya et al. refer to these Arabidopsis genes as AtCHL1 and AtCHL2, respectively. Expression of CaCLH, AtCLH1, and AtCLH2 in E. coli resulted in chlorophyllase activity (Tsuchiya et al., supra).
The CaCLH deduced amino acid sequence has between 32% and 40% sequence homology with that of AtCHL1 and AtCHL2. All three polypeptides have highly conserved regions that correspond to a conserved motif present in several bacterial, animal, and plant lipases and hydrolases which includes a potential ATP/GTP-binding-site motif, or P-loop. Homology of the entire polypeptide of any of the chlorophyllases with other known lipases is less than 10%.
AtCHL2 was originally labeled COI1 and was isolated from an Arabidopsis thaliana mutant (coi1, for coronatine-insensitive 1) which is insensitive to methyl jasmonate (MeJA) and coronatine and produces sterile male flowers. AtCHL1 was originally labeled ATHCOR1 (for Arabidopsis thaliana coronatine induced) and was identified as being induced by MeJA, coronatine, and wounding (Benedetti et al. (1998) Plant Physiol. 116:1037–1042). Coronatine is a phytotoxin produced by some plant-pathogenic bacteria, and mimics the action of MeJA in plants. MeJA is a plant-signaling molecule involved in stress responses such as wounding and pathogen attack and is essential for pollen grain development in Arabidopsis thaliana. 
Because of the involvement of chlorophyll degradation in plant cell senescence, identification of cDNAs encoding chlorophyllases in other plants, particularly economically important crop plants, will allow the controlled induction or postponement of senescence.